Nattokinase (NK) is a potent fibrinolytic enzyme with wide pharmaceutical and nutraceutical applications. Safe and high NK-yielding strains are urgently needed. In this study, the best strain NDF was isolated from one of the 11 natto samples and then identified as Bacillus subtilis. The effects of carbon and nitrogen sources on NK production were investigated, and glucose and soybean milk were finally selected as the optimal carbon and nitrogen sources, respectively. Acetoin, a valuable compound with versatile usages, was detected as the main byproduct of carbon overflow. In a 6-L fermenter, NK and acetoin reached their peak concentrations simultaneously (10,220 IU/mL and 25.9 g/L, respectively) at 25 h in a culture medium containing 180 g/L of soybean milk and 105 g/L of glucose. The NK product was verified by sequencing of the aprN gene and SDS-PAGE analysis. Only very limited kinds of proteins were found in the supernatant of the fermentation broth, and NK was one of the main bands. This study has developed an economical and high NK production method with acetoin as a useful byproduct.

纳豆激酶 (NK) 是一种有效的纤溶酶，具有广泛的制药和营养保健用途。迫切需要安全且高 NK 产量的菌株。本研究从11个纳豆样品中分离出最佳菌株NDF，并鉴定为枯草芽孢杆菌。考察了碳源和氮源对NK产生的影响，最终选择葡萄糖和豆浆分别作为最佳碳源和氮源。乙偶姻是一种具有多种用途的有价值的化合物，被检测为碳溢流的主要副产品。在 6 L 发酵罐中，在含有 180 g/L 豆奶和 105 g/L 葡萄糖的培养基中，NK 和乙偶姻在 25 小时同时达到峰值浓度（分别为 10,220 IU/mL 和 25.9 g/L） 。通过aprN基因测序和 SDS-PAGE 分析验证 NK 产物。发酵液上清液中仅发现非常有限的蛋白质种类，NK是主要条带之一。本研究开发了一种经济高效的 NK 生产方法，以乙偶姻作为有用的副产物。